Patterns of Antibiotic Resistance in Enterobacteriaceae Isolates from Broiler Chicken in the West Region of Cameroon: A Cross-Sectional Study

Background The emergence of multidrug-resistant food-borne pathogens of animal origin including Enterobacteriaceae is a growing concern. Identifying and monitoring resistance in isolates from human-related environments are of clinical and epidemiological significance in containing antimicrobial resistance. This study aimed to contribute towards the fight against antibiotic resistance and ameliorate the management/treatment of Enterobacteriaceae-linked diseases in Cameroon. Methods Cloacal swabs from healthy broilers were enriched in buffered-peptone-water and cultured on EMB agar. Antibiotic susceptibility was tested on Mueller-Hinton-Agar by disc diffusion. Plasmid-borne genes for extended-spectrum beta lactamase (ESBL) and resistance to Quinolones (PMQR) and Aminoglycosides were detected by standard endpoint polymerase chain reaction (PCR). Results A total of 394 isolates were identified belonging to 12 Enterobacteriaceae genera, the most prevalent were Escherichia coli (81/394 = 20.56%), Salmonella spp (74/394 = 18.78%), and Klebsiella spp (39/394 = 9.90%) respectively. Overall, 84/394 (21.32%) were ESBL producers, 164/394 (41.62%) were resistant to quinolones, 66/394 (16.75%) resistant to aminoglycosides with 44.0% (173/394) expressing MDR phenotype. Poor hygiene practice (OR 2.55, 95% CI: 1.67, 3.89, p=0.001) and rearing for >45 days, (OR = 7.98, 95% CI: 5.05, 12.6, p=0.001) were associated with increased carriage of MDR. Plasmid-borne resistance genes were detected in 76/84 (90.48%) of ESBL-producing isolates, 151/164 (92.07%) quinolone resistant isolates and 59/66 (89.39%) aminoglycoside resistant isolates with co-occurrence of two or more genes per isolate in 58/84 (69.05%) of ESBLs, 132/164 (80.49%) of quinolone resistant isolates and 28/66 (42.42%) of aminoglycoside resistant isolates. Conclusion This study found high carriage and widespread distribution of Enterobacteriaceae with ESBL and MDR in broiler chicken in the West Region of Cameroon. Most PMQR genes in bacteria were found at levels higher than is seen elsewhere, representing a risk in the wider human community.


Introduction
Enterobacteriaceae is a heterogeneous Family of Gram negative non-spore forming bacilli which primarily live in guts of humans and other higher animals where they can be pathogens or members of the normal intestinal microflora but are also widely distributed in soil, water, and decaying matter [1,2]. Many members of this Family are clinically important pathogens with extensive capacity to develop antibiotic resistance (ABR) due to a myriad of plasmidborne resistance genes and a high capacity of mutation in the phase of environmental stress such as in the presence of antibiotics [3][4][5].
ese have made Enterobacteriaceae a serious threat to public health due to the association of different resistance mechanisms and the insufficient development of new drugs which make these microorganisms resistant to almost all available antibiotics [6].
e World Bank multiple indicators cluster survey (MICS5), 2014 shows that in Cameroon, diarrheal diseases (Enterobacteriaceae being a main cause) account for 5,01% of annual deaths; being the 5th cause of mortality after malaria, HIV-AIDS, neonatal diseases, and lower respiratory diseases [7]. e country's present antimicrobial resistance (AMR) surveillance targets only priority diseases including malaria, HIV-AIDS, and tuberculosis but the development of multidrug resistance (MDR) in Enterobacteriaceae as reported in local studies on human and animal health and the environment is acknowledged by the Ministry of Health [8] with among others, an MDR of 39.8% in Salmonella serotypes from chicken in retail markets in Yaoundé gotten by Wouafo et al. [9] and an increase in the resistance by E. coli to reference antibiotics against Enterobacteriaceae including cephalosporins, fluoroquinolones and aminoglycosides within the period from 2009 to 2013 noted by Longla et al. at the Yaoundé University Teaching Hospital [10]. However, the National Antimicrobial Resistance Action Plan 2018-2020 envisaged the surveillance of pathogenic Enterobacteriaceae based on WHO criteria of timely detection, reporting, risk assessment and monitoring of emerging ABR including Shigella spp, Salmonella spp, E. coli and K. pneumoniae [8]. It is thus of epidemiological importance to monitor the trends of resistance in these bacteria isolated from human-related environment such as food animals so as to contain the vicious cycle whereby human activities induce the development of ABR in bacteria which in turn have repercussions on human health [11]. e area of study, the West Region of Cameroon is intensively involved in animal husbandry, mainly poultry and pig farming which provide food and jobs to a great number of people; these activities highly linked to foodborne infections [12,13]. We focus on chicken, an important source of contamination with clinically important human pathogens [14] and a main source of meat for households and mass catering events in the Region known for its attachment to cultural celebrations including burial and funeral celebrations [15]. ey tend to harbour extensive numbers and diverse types of enteric bacteria both commensal and pathogenic [16]. Such co-occurrence coupled with abuse of antibiotics in Cameroonian poultries [17] can give opportunity for sharing of resistance genes among species by means of mobile genetic elements (MGEs) and chromosomal recombination due to stress from antibiotics leading to emergence of resistant and multidrug resistant strains [3,4,16].
When AMR develops in commensal intestinal microflora it tends to go unchecked making these commensals to act as reservoir of AMR [16]. us the development of AMR in commensal Enterobacteriaceae of animal origin is a measure for early detection of AMR in the community [18].
ere is therefore need for thorough research on AMR as a contribution to the country's development plans. e few studies carried out in the West Region have been centred on human isolates and clinically important Enterobacteriaceae and also at point locations [19][20][21]. With the lack of epidemiological data on prevalence and ABR among Enterobacteriaceae, pathogens and commensals alike in humanrelated environment, chicken being one of the main meat types in Cameroon [22] and an important source of Enterobacteriaceae infection for humans [14], and bearing in mind that the development of ABR in commensal Enterobacteriaceae of animal origin is a measure for early detection of ABR in the community [18], our quest for patterns of ABR in Enterobacteriaceae in the West Region of Cameroon prompted us to use chicken as our sample source.
is research aimed to uncover the extent of the problem of antimicrobial resistance and its risk factors among poultry farming community in West region, noted as the production bay in Cameroon. It is hoped that information gathered would call the attention of stakeholders, including veterinarians, physicians, microbiologists, livestock producers, public health workers and relevant government agencies to the need for basic salvaging measures to curb the development and dissemination of ABR and contribute towards the management of Enterobacteriaceae-linked diseases.

Aim.
e aim of this study was to contribute towards the fight against the development of ABR so as to ameliorate the management and treatment of diseases linked to Enterobacteriaceae in Cameroon; providing epidemiological data on prevailing Enterobacteriaceae and their resistance patterns and risk factors.

Study Design.
e study was a cross-sectional study which ran from October 2018 to September 2021, punctuated from January 2020 to January 2021 due to the COVID-19 pandemic. October 2018 to December 2020 was field work and sampling, laboratory isolation and characterisation, and antibiotic susceptibility testing. February 2021 to September 2021 was molecular analysis of antibiotic resistance genes.

Study Site.
e West Region with an area of 13,892 km 2 is a territory located in the central-western portion of the Republic of Cameroon located at 5°30′N 10°30′E. It shares borders with the North West Region to the northwest, the Adamaoua Region to the northeast, the Centre Region to the southeast, the Littoral Region to the southwest, and the Southwest Region to the west. e West Region is the smallest of Cameroon's ten regions in area, yet it has the second highest population density; a population of 1,865,394 (2013) with density of 142.9 inhabitants/km 2 as of 2017 [23,24]. Its Capital is Bafoussam in the Mifi Division. It has 8 Divisions: Bamboutos, Upper-Nkam, Upper-Plateau, Koung-Khi, Ménoua, Mifi, Ndé and Noun [24]. e Region is found in the Grass field plateaus of the Western Highlands with a cold climate and the main ethnic groups are the Bameliké and Bamoum [25]. Figure 1 below shows the localisation of the study site. Laboratory work was carried out at the Research Unit of Microbiology and Antimicrobial Substances (RUMAS) at the University of Dschang.

Sample Collection.
e study was performed on cloacal swabs from healthy broiler chicken irrespective of age from 28 farms in 11 locations in 5 out of the 8 Divisions of the West Region of Cameroon. We used stratified sampling with Divisions as the strata.
Cloacal swabs were collected by inserting a sterile swab into the cloaca. Each swab was immersed in 5 ml of a sterile physiological solution in swab sampling tubes. e samples were transported in cool thermo-flasks to the laboratory for analysis [26].

Sample Enrichment and Culture.
e swabs were dissolved in 10 ml of buffered peptone water and incubated for 24 hrs at 37°C. e enriched samples were cultured on EMB agar by plate streaking and incubated for 48 hours [1,27].

Isolation of Isolates and Preservation.
Isolated colonies on the EMB agar were identified based on colony characteristics, picked and conserved in a conservation medium; a mixture of glycerol and Muller Hilton broth at 1 part to 3 parts and stored in a refrigerator at − 20°C [28,29].

Phenotypic Characterisation of Isolates.
Isolates were identified using colony characteristics on the culture medium (EMB agar) and 10 rapid screening biochemical tests [1,30] locally used in routine clinical laboratory diagnosis including: glucose and lactose fermentation, hydrogen sulphide (H 2 S) and gas production using Kligler Iron Agar (KIA), urease activity and indole production using urea broth and Kovacs' reagent, mannitol fermentation and motility using mannitol agar, citrate fermentation using citrate agar and catalase test.

Antibiotic Susceptibility
Testing. Fresh colonies were used to prepare 0.5 McFarland solution inoculums using sterile physiological water. Antibiotic susceptibility testing was carried out on Mueller Hilton Agar by the Kirby-Bauer disc diffusion method [2,30]. Screening for ABR in the isolates was done using the following antibiotics in Table 1 [31], and resistant control strains (ATCC stains of E. coli, K. pneumoniae and Salmonella Typhi) maintained by the pharmacology research group of the RUMAS.
Phenotypic determination of resistance by penicillinase production was tested by combination disc test with amoxicillin/clavulanic acid with comparison to amoxicillin. Extended spectrum beta-lactamase production was tested by observing resistance to amoxicillin followed by resistance to ceftriaxone or cefotaxime [32]. Confirmation of ESBL production was done by double disc synergy test on MHA by placing a disk of amoxicillin-clavulanic acid at the centre surrounded by discs of cefotaxime, ceftriaxone and ceftazidime 2 cm apart and observing for a clear zone of intersection between the central and peripheral discs [33,34].

Detection of Resistance
Genes. DNA extraction from fresh colonies was done by heat shock. A loop-full of a fresh colony was dissolved in 400 μl of Tris-EDTA 1X buffer (Tris-Cl 0.1 M and EDTA 0.01 M diluted 1/10). e solution was vortexed for 5 s and heated in a water bath at 95°C for 25 min. e heated solution was centrifuged at 13000 tpm for 3 min and the supernatant containing DNA was extracted and used for PCR [35].
Amplification of some representative epidemiologically important plasmid-borne resistance genes including ESBL genes, PMQR genes and plasmid-mediated aminoglycoside resistance (PMAR) genes was done using the following oligonucleotides and reaction conditions in Table 2: PCR was done with a 25 μl reaction mix composed of 14.9 μL of PCR grade water, 2.5 μL of 1X standard Taq buffer solution with 2.5 mM MgCl2, 1 μL of forward primer, 1 uL of reverse primer, 0.5 μL of DNTP mix, 0.1 μL of standard Taq and 5 μL of DNA solution in a TECHNE ® thermocycler [40].
Reaction products were migrated on a 1.5% agarose gel and revealed under UV light.

Data Analysis.
is research work generated information on Enterobacteriaceae carriage in broilers, identification of Enterobacteriaceae organisms, their prevalence, their antibiotic susceptibility profiles (susceptible, resistant, multidrug resistant and ESBL producing) and, odds ratios and correlations between outcomes (Enterobacteriaceae carriage, prevalence, resistance, MDR and ESBL production) and risks.
Enterobacteriaceae carriage was determined by the count of the different colonies on the Enterobacteriaceae-specific growth medium (EMB agar) based on colony aspect and morphology. e identity of the organism was gotten by interpreting the colony aspect and the results of the phenotypic reactions using the table of reactions below in Table 3 [41][42][43][44].
Susceptibility to antibiotics was evaluated using standard values as given by Sigma Aldrich ® [45]. MDR was assessed as resistance of an isolate to two or more antibiotics belonging antibiotic classes of choice used against Enterobacteriaceae (cephalosporins, carbapenems, quinolones or aminoglycosides). ESBL production was interpreted from double disc synergy test between amoxicillin/clavulanic acid disc and ceftriaxone, ceftazidime or cefotaxime disc.
Arithmetic operations and conversions were done using Microsoft Excel sheets while frequencies, prevalence, odds ratios, correlations and diagrams were done using IBM SPSS Statistics 20.

Evaluation of Risks in Farms.
Samples were collected from 28 farms in 11 locations in 5 out of the 8 Divisions in the West Region. Evaluating risks, 25 farms out of 28 (89.29%) used antibiotics on their chicken, 138/275 (50.18%) subjects sampled were more than 30 days old and fell on the category "old" while 137/275 (49.18%) were 30 days old or less and fell in the category "young", 7 farms out of 28 (25.00%) regularly cleaned feeders and drinkers for the chicken, 12 farms out of 28 (42.86%) gave unsure water to their chicken and 18 out of 28 farms (64.29%) had dirty environment with or without stagnant sewage. Table 4 below gives information on the locations of farms, number of samples and isolates and the risk factors to which chicken subjects were exposed.
A total of 275 cloacal swabs were collected. All these samples tested positive for Enterobacteriaceae with a carriage of at least 2 different colony types and a mean of 3 different colony types per sample. Figure 2 below shows a bacterial carriage of 6 different colony types in one sample.
Bacterial carriage of greater than or equal to 3 different colony types present in culture correlated significantly with the 3 environmental risk factors evaluated as shown in Table 5 below.

Prevalence of Members of the Enterobacteriaceae.
From the 275 cloacal swabs collected, a total, 394 isolates were obtained and characterised belonging to 12 Figure 3 below. Detailed information on the prevalence at each sampling site is given in Table 6 below as percentages only to render the information less cumbersome. e computed coefficient of variation of the prevalence of each organism in the various locations showed relatively uniform prevalence across the study area.  Farkas, et al. [36]. Minh Vien et al. [38].

Prevalence of Antibiotic
Kim, et al. [39]. ere were 230 (58.38%) of isolates resistant to at least one antibiotic class of choice against Enterobacteriaceae (cephalosporins, carbapenems, quinolones or aminoglycosides). 80 (20.3%) of the isolates were resistant to at least one of the cephalosporins tested, 164 (41.62%) were resistant to at least one of the quinolones tested and 66 (16.75%) were resistant to at least one of the aminoglycosides tested. 232 (58.88%) of the isolates showed penicillinase production but ESBL production was much lower with 84 (21.32%) and 173 (44.0%) showed MDR when considering only beta lactams, quinolones and aminoglycosides, but adding the contribution of tetracyclines MDR rose to 85.53% (337 isolates). Table 7 below gives details of the prevalence of ABR in various isolates given as percentages only to make the information less cumbersome. e overall prevalence of resistance and MDR are presented in Figures 4and 5 below. e association of resistance phenotypes to the various antibiotic classes is shown in Figure 6 below. Figure 6 shows that 230 out of 394 isolates (58.38%) were at least either ESBL producer, quinolone resistant or aminoglycoside resistant, an association of two or of all the 3 resistances studied. It also shows that the number of ESBL producing isolates that were quinolone resistant was Table 4: Locations of farms, number of samples and isolates and the risk factors to which chicken subjects were exposed. Using any other source of water apart from pipe-borne water without prior treatment. Use of antibiotics Use of antibiotics in feed for chicken subjects.

Sanitation
Conditions of the environment such as litter, stagnant sewage, rearing of animals around poultry farm, state of workers' restroom.

Feeding hygiene
Cleaning of feeders and drinkers at least twice a week. Age ≤30 days for category "young" and ˃30 days for category "old" chicken.
Bacterial carriage per sample.
Large, pink and mucoid

Citrobacter
Small, flat, dark violet with or without a metallic green sheen in reflected light Large, transparent and circular

Association of Risk Factors among Chickens with Antibiotic
Resistance. e outcome "resistance" indicates isolates showing resistance to at least one antibiotic from one class of choice. e development of resistance to at least one antibiotic class correlated significantly to the age of the chicken (p � 0.001, OR � 13.491) and food hygiene (p � 0.007, OR � 1.783). Isolates had higher risk of developing resistance on exposure to all risks except unsure water. ESBL production correlated significantly to age of chicken (p � 0.001, OR � 4.505) and isolates had higher risk of developing ESBL on exposure to all risks except use of antibiotics. MDR correlated significantly to the age of the chicken (p � 0.001, OR � 7.980) and isolates had a higher risk of developing MDR on exposure to all risks except unsure water. e risk "use of antibiotics" was quasi constant thus did not correlate with outcomes but increased the chances of developing the various outcomes as seen in the OR greater than 1. e association of risks to resistance outcomes is presented in Table 8 below.

Detection of Plasmid-borne Resistance Genes in Isolates.
e prevalence of some representative clinical and epidemiologically important plasmid-borne genes in the phenotypically resistant isolates as detected by PCR are presented per resistance to each antibiotic class in the subsections below. Added are the details about 3 most prevalent and local clinically important genera; E. coli, Salmonella and Klebsiella. Worth noting here also is the relatively high occurrence of the resistance genes in clinically less important isolates under the group "Others".

Detection of Plasmid-borne Beta Lactamase Genes.
Six beta lactamase genes were amplified as shown in Table 9 below. e blaTEM-1 gene was the most prevalent gene among beta lactamase producers in 59/84 (70.24%) of the isolates. Figure 7 below is a sample gel image showing the amplification of gene fragment of the blaTEM-1 gene, one of the beta lactamase genes amplified by PCR.

Detection of PMQR Genes.
Five PMQR genes were amplified as shown in Table 10 below. e aac (6′)-IB-CR, a variant of the aminoglycoside resistance gene that confers resistance to quinolones was the most prevalent gene among PMQR genes in 97/164 (59.15%) of the isolates. Figure 8 below is a sample gel image showing the amplification of a fragment of the qnrS gene, one of the PMQR genes amplified by PCR.

Detection of PMAR Genes.
ree PMAR genes were amplified as shown in Table 11 below. e aac(6′)-IB gene was the most prevalent gene among PMAR genes in 51/66 (77.27%) of the isolates.

Co-occurrence of Plasmid-borne Resistance Genes
Enterobacteriaceae.
e Co-occurrence of the Various Plasmid-borne Genes as Detected by PCR Is Summarized in Table 12 below.

Discussion
In this study, we sought to determine the extent and distribution of antimicrobial resistance phenotypes and detect plasmid-mediated genes associated with ESBL production, Quinolone and Aminoglycoside resistance among broilers in poultry farms in the West region of Cameroon. We also identified risk factors that were associated with the carriage of multidrug resistant forms of the isolates across the study area.
In total, 394 isolates were detected in all samples analysed. ere was a high prevalence of the genera Escherichia 81/394 (20.56%) and Salmonella 74/394 (18.78%) among the isolates followed by Klebsiella 39/394 (9.90%). ese genera have clinically important species causing various diseases in human [46] thus the need to monitor ABR in these bacteria. e isolation of members such as Shigella even in few numbers is already a cause for concern given their potential to cause epidemics when virulent. However, the prevalence is similar to what is found elsewhere in several studies [47,48]. e fairly uniform prevalence of the organisms in locations studied can be explained by the uniform sociodemographic and geographical nature (grass field highlands) of the area [25].
Overall, the Enterobacteriaceae organisms isolated showed a high level of resistance to amoxicillin 345/394 (87.8%) of the group of Penicillins and doxycycline 380/ 394 (96.7%) of the class of Tetracyclines. ese results were concordant with results obtained in 2013 by Tago et al. in Ivory Coast, a similar geographic area to Cameroon [48] and further confirmed that these are no more drug classes of choice against Enterobacteriaceae. Traditionally Enterobacteriaceae have developed high resistance to Penicillins via production of penicillinase [49] this explains high resistance to amoxicillin. As seen in similar studies, E. coli and Klebsiella maintained their known high production of ESBLs [50] [51], resistance to quinolones is on the rise with for example ciprofloxacin 10% (90%  parts of Cameroon owing to rampant drug misuse and self-medication which is not only in clinical settings but also in poultry farms [17,19,20,52]. Multidrug resistance was observed to be high in all the genera with an overall prevalence of 337/394 (85.53%). is is similar to the trend seen elsewhere as reported by Leski   ESBL production was observed at fairly moderate levels but the co-development of MDR and ESBL further compounds the failure of antibiotics in disease treatment [56]. A bacterial carriage of an average of 3 different Enterobacteriaceae colony types was found in each sample indicating the possibility of the development of ABR and MDR through horizontal gene transfer by means of MGEs such as plasmids [17,30].   is research showed from the analysis of the odds ratios of risks that poor sanitation at poultry farm, from environment to feeding predisposed chicken first to high bacterial carriage which predisposed these bacteria to developing resistance and MDR. e sanitary conditions in the poultries visited were average, not up to standard conditions mainly due to the nature of construction and materials used which promoted poor hygiene around the poultry farms. Some areas did not have pipe borne water thus farmers used well water with doubtful cleanliness. ese are conditions that usually favour extensive infection of the animals [57,58] and this was confirmed by observations made in the current study which found an increase in the odds of infection of chicken among farms with poor feeding hygiene practice and poor sanitary practice in poultry farm.
ough not significant, antibiotic use was seen to be a risk factor with OR 1.40. is is because almost all poultry farms 26/28 (92.86%) used antibiotics regularly making it a statistical constant, as reported by Guetiya et al. [52]. Correlation of these outcomes with long rearing periods (>45 days) could be explained by the long duration of exposure to the risks [59], the time to get infected or for coinfection to allow horizontal gene transfers.   is research also showed that ESBL producing isolates that were quinolone resistant were significantly lower than those that were not quinolone resistant (32/84 against 52/84 respectively) while aminoglycoside isolates that were resistant to quinolones were significantly higher than those that were not quinolone resistant (43/66 against 23/66). is suggests that quinolone resistance could not be largely plasmid-mediated because plasmid-carried quinolone resistance genes tend to occur with ESBL producing genes creating the opposite scenario [56]. is observation thus suggests other mechanisms of resistance such as a chromosomal DNA based quinolone resistance could be participating due to antibiotic misuse. López et al. showed that ciprofloxacin induced chromosomal recombinations in E. coli that enabled the bacterium to resist the drug [4]. However, given the high association of aminoglycoside resistance to quinolone resistance, it suggests a shared rather than intrinsic mechanism such as plasmid-mediated resistance but the lower overall prevalence of resistance to aminoglycosides shows a smaller contribution of this mechanism in the resistance observed [60,61].
In this study, we detected plasmid-borne resistance genes to all the three drug classes of choice used against Enterobacteriaceae-beta lactams, quinolones and aminoglycosides.
ere was a high co-occurrence of several resistance genes in the isolates probably indicating the gravity of dissemination of these genes. e case where some isolates tested negative for all the genes tested [in 8/84 (9.5%) ESBL producing isolates, 13/164 (7.9%) quinolones resistant isolates and 7/66 (10.6%) aminoglycosides resistant isolates] can be explained in two ways: first, it may be that the plasmid-borne resistance gene responsible for the resistance phenotype was not tested since the genes tested were not exhaustive; second, it may be that other genetic mechanisms than plasmid mediation such as chromosomal encoded resistance and chromosomal mutations may be responsible [3,4].
We observed a high prevalence of the beta lactamase genes blaTEM1 59/84 (70.24%), blaCTX-M 19/84 (22.62%) and blaKPC 30/84 (35.71%) of the TEM, CTX and KPC enzyme families in isolates. ese are genes for Class A beta lactamase enzymes which are highly disseminated among important Enterobacteriaceae pathogens. is is an indication of the risk of their further dissemination and the resistance they confer owing to their occurrence in MGEs and also their ability to expand their spectrum of activity as new antibiotics are developed especially by shuffling of chromosomal genes and mutations [62].
ese are clinically important genes to monitor in epidemiological studies because of their enrichment in human-associated environments, mobility, and presence in pathogens [63]. e high occurrence of the aac(6′)-IB-CR gene 97/164 (59.15%), though a low level mediator of resistance to ciprofloxacin [64] [67].
ough Zhang et al. classified the qnrS and qepA as low risk gene due to their low dissemination in clinical and humanrelated environment [63], we note in this study that these genes show a high enrichment in the community studied and correlates with the high resistance to quinolones observed in this study. us with this relatively high prevalence they also need to be carefully monitored. PMQR genes showed a high co-occurrence with 132/164 (80.49%) of the isolates positive for more than one PMQR gene, a further indication of their high dissemination. Of the three enzyme classes: N-Acetyltransferases (AAC), O-Adenyltransferases (ANT) and O-Phosphotransferases (APH) involved with PMAR, the aac(6′)-IB gene coding for an N-acetyltransferase is the most clinically important gene to monitor in epidemiological studies because of its enrichment in human-associated environments, mobility, and presence in pathogens [63,68]. is gene had the highest occurrence 51/ 66 (77.27%) in the isolates tested and this correlates with the high occurrence of its variant, the aac(6′)-IB-CR gene 97/164 (59.15%), responsible for PMQR. However, the aph(3′)-IA and the ant(2′)-IA genes considered non-epidemiologically important because of their previous low enrichment in human-related environment and pathogens [63] showed a much higher prevalence of 23/66 (34.85%) for aph(3′)-IA and 14/66 (21.21%) for ant(2′)-IA. is indicates that these genes are gradually being enriched in the community and there is need to curb their further dissemination. ese ESBL, PMQR and PMAR genes have also been isolated from human pathogens showing a generalised circulation between humans and animals [33,53,56,69].
is lays emphasis on the need for the extension of the fight against ABR to animal husbandry in Cameroon.
Molecular methods such as the DNA microarray have been developed to simultaneously detect microbes in pathologic samples [70]. It would no doubt have been a powerful tool in our research to detect bacteria with a lot of precision. However, the application of such tools may be limited by cost. Furthermore, determining the different strains of important bacteria with a much higher resolution technique such as the use of Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) genotyping [71] would have thrown more light into the strains contributing to much of the observed resistance.
is will form part of future studies alongside the hypotheses developed through the present investigations.

Conclusion
is study showed a high carriage of Enterobacteriaceae showing phenotypic resistance with corresponding plasmidborne resistance genes, a widespread and a fairly uniform distribution among broilers in poultry farms across the study area. Enterobacteriaceae from chicken in this Region showed high resistance to Penicillins and Tetracyclines and low resistance rates to 3rd generation Cephalosporins and Aminoglycosides. Poor hygienic conditions at poultry farms and rearing chicken for long periods were associated with increased carriage of multi drug resistant Enterobacteriaceae. Plasmid-mediated genes for ESBL production, quinolone resistance and aminoglycoside resistance were extensively distributed in both pathogenic and commensal Enterobacteriaceae with high co-occurrence of the genes in the isolates. e high prevalence of MDR especially in clinically important genera like Salmonella, Escherichia and Klebsiella indicates the necessity for continuous monitoring and for stakeholders to put efforts and resources to improve sanitation at poultry farms and combat resistance development and misuse of antibiotics in animal farms.

Data Availability
e numerical data used to support the findings of this study are included in the article.

Ethical Approval
is study did not involve active participation of the fowls and sampling by cloacal swabs was a non-invasive procedure. However, working with poultry farms falls under the authority of the Ministry of Livestock, Fisheries and Animal Industries (MINEPIA in its French acronym), thus an authorisation (authorisation reference number 68/18/L/ DREPIA-O/SRAG of 04/06/2018) to sample chicken within the West Region was obtained from the Regional Delegation of MINEPIA which also helped to enable collaboration with its Divisional Delegates and farmers.

Consent
Consent by poultry farm owners to sample broilers was verbal after presentation of researcher credentials, research authorisation and explanation of the work and the sampling procedure.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.